Detection of fusarium species infecting corn using the polymerase chain reaction

ABSTRACT

The present invention relates to the use of primers in polymerase chain reaction assays for the detection of a  Fusarium proliferatum, F. verticillioides  and  F subglutinans . Specific primers are identified as being useful for the identification of fungal isolates using PCR based techniques.

FIELD OF THE INVENTION

[0001] The present invention relates to the use of primers in polymerasechain reaction assays for the detection of maize Fusarium ear rotpathogens Fusarium subglutinans, F. proliferatum, and F. verticillioides(syn. F. moniliforme). The use of these primers enables the detection ofspecific isolates of fungal pathogens and the monitoring of diseasedevelopment in plant populations.

BACKGROUND OF THE INVENTION

[0002] Diseases in plants cause considerable crop loss from year to yearresulting both in economic deprivation to farmers and, in many parts ofthe world, to shortfalls in the nutritional provision for localpopulations. The widespread use of fungicides has provided considerablesecurity against plant pathogen attack; however, despite $1 billionworth of expenditure on fungicides, worldwide crop losses amounted toapproximately 10% of crop value in 1981 (James, 1981, Seed Sci. &Technol. 9: 679-685).

[0003] The severity of the destructive process of disease depends on theaggressiveness of the pathogen and the response of the host. One aim ofmost plant breeding programs is to increase the resistance of hostplants to disease. Typically, different races of pathogens interact withdifferent varieties of the same crop species differentially, and manysources of host resistance only protect against specific pathogen races.Furthermore, some pathogen races show early signs of disease symptoms,but cause little damage to the crop. Jones and Clifford (1983, CerealDiseases, John Wiley) report that virulent forms of the pathogen areexpected to emerge in the pathogen population in response to theintroduction of resistance into host cultivars and that it is thereforenecessary to monitor pathogen populations. In addition, there areseveral documented cases of the evolution of fungal strains that areresistant to particular fungicides. As early as 1981, Fletcher and Wolfe(1981, Proc. 1981 Brit. Crop Prot. Conf.) contended that 24% of thepowdery mildew populations from spring barley and 53% from winter barleyshowed considerable variation in response to the fungicide triadimenoland that the distribution of these populations varied between varieties,with the most susceptible variety also giving the highest incidence ofless susceptible types. Similar variation in the sensitivity of fungi tofungicides has been documented for wheat mildew (also to triadimenol),Botrytis (to benomyl), Pyrenophora (to organomercury),Pseudocercosporella (to MBC-type fungicides) and Mycosphaerellafijiensis to triazoles to mention just a few (Jones and Clifford, CerealDiseases, John Wiley, 1983).

[0004] Maize Fusarium ear rots are caused by Fusarium verticillioides,F. proliferatum, and F. subglutinans. The importance of the disease isderived from the production of the mycotoxin fumonisin by the causalorganisms (Compendium of Corn Diseases, 3^(rd) ed., D. White Ed., APSPress, 1999). Contaminated grain can cause serious problems for themaize feed and food industries (Munkvold and Desjardins, 1997, PlantDisease 81(6):556-565). Fumonisins inhibit the biosynthesis ofsphingolipids, changing the sphingolipid composition of a number oftarget tissues, and can cause a variety of diseases in animals that eatcontaminated feeds (Munkvold and Desjardins, 1997). Consumption of maizecontaminated with high levels of fumonisins has been epidemiologicallyassociated with high levels of esophageal cancer in human populations inparts of the world where maize is a staple food (Munkvold andDesjardins, 1997). This situation is further complicated by the commonoccurrence of fumonisins in symptomless infected kernels (Desjardins andPlattner, 1998, Plant Disease 82(8):953-958). Though Fusarium ear rotstypically do not significantly affect yield, they do introducemycotoxins to the grain, leading to the loss of grain and seed quality.

[0005] In view of the above, there is a real need for the development oftechnology that will allow the identification of specific races ofpathogen fungi early in the infection process. By identifying thespecific race of a pathogen before disease symptoms become evident inthe crop stand, the agriculturist can assess the likely effects offurther development of the pathogen in the crop variety in which it hasbeen identified and can choose an appropriate fungicide if suchapplication is deemed necessary.

SUMMARY OF THE INVENTION

[0006] The present invention is drawn to methods of identification ofdifferent pathotypes of plant pathogenic fungi. The invention providesprimers derived from either the mitochondrial Small Subunit RibosomalDNA sequences or Internal Transcribed Spacer (ITS) sequences of thenuclear ribosomal RNA gene (rDNA) of different fungal pathotypes. Theseprimers generate unique fragments in PCR reactions in which the DNAtemplate is provided by specific fungal pathotypes and can thus be usedto identify the presence or absence of specific pathotypes in host plantmaterial before the onset of disease symptoms.

[0007] In a preferred embodiment, the invention provides diagnosticprimers from Mitochondrial Small Subunit (SSU) rDNA or the InternalTranscribed Spacer (ITS) sequences of the nuclear ribosomal RNA gene forthe detection of Fusarium subglutinans, F. proliferatum, and F.verticillioides.

[0008] This invention provides the possibility of assessing potentialdamage in a specific crop variety-pathogen strain relationship and ofutilizing judiciously the diverse armory of fungicides that isavailable. Furthermore, the invention can be used to provide detailedinformation on the development and spread of specific pathogen racesover extended geographical areas. The invention provides a method ofdetection that is especially suitable for diseases with a long latentphase.

[0009] Kits useful in the practice of the invention are also provided.The kits find particular use in the identification of Fusariumsubglutinans, F. proliferatum, and F. verticillioides.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

[0010] SEQ ID NO:1 Fusarium verticillioides (syn. F. moniliforme) smallsubunit ribosomal RNA, mitochondrial gene encoding mitochondrial RNA,partial sequence. GenBank Accession Number U34497.

[0011] SEQ ID NO:2 Fusarium proliferatum NRRL 22944 small subunitribosomal RNA, mitochondrial gene encoding mitochondrial RNA, partialsequence. GenBank Accession Number U34500.

[0012] SEQ ID NO:3 Gibberella zeae (syn. Fusarium graminearum) smallsubunit ribosomal RNA, mitochondrial gene encoding mitochondrial RNA,partial sequence. GenBank Accession Number U34520.

[0013] SEQ ID NO:4 Fusarium subglutinans small subunit ribosomal RNA,mitochondrial gene encoding mitochondrial RNA, partial sequence. GenBankAccession Number U34501.

[0014] SEQ ID NO:5 Fusarium subglutinans internal transcribed spacerRNA. GenBank Accession Number U34559.

[0015] SEQ ID NO:6 Gibberella zeae NRRL 5883 internal transcribed spacerRNA. GenBank Accession Number U34578.

[0016] SEQ ID NO:7 Fusarium proliferatum NRRL 22944 internal transcribedspacer RNA. GenBank Accession Number U34558.

[0017] SEQ ID NO:8 Fusarium verticillioides (syn. F. moniliforme)internal transcribed spacer RNA. GenBank Accession Number U34555.

[0018] SEQ ID NO:9 Oligonucleotide Primer ITS1

[0019] SEQ ID NO: 10 Oligonucleotide Primer ITS2

[0020] SEQ ID NO: 11 Oligonucleotide Primer ITS3

[0021] SEQ ID NO: 12 Oligonucleotide Primer ITS4

[0022] SEQ ID NO: 13 Oligonucleotide Primer FCORN1

[0023] SEQ ID NO: 14 Oligonucleotide Primer FCORN2

[0024] SEQ ID NO: 15 Oligonucleotide Primer FSUB1

[0025] SEQ ID NO: 16 Oligonucleotide Primer FSUB2

[0026] SEQ ID NO: 17 Oligonucleotide Primer FSUB3

[0027] SEQ ID NO: 18 Oligonucleotide Primer FVERT1

[0028] SEQ ID NO: 19 Oligonucleotide Primer FVERT2

[0029] SEQ ID NO:20 Oligonucleotide Primer FPRO1

[0030] SEQ ID NO:21 Oligonucleotide Primer FPRO2

[0031] SEQ ID NO:22 Oligonucleotide Primer FPRO3

[0032] SEQ ID NO:23 Oligonucleotide Primer MS1

[0033] SEQ ID NO:24 Oligonucleotide Primer MS2

DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention provides unique DNA sequences that areuseful in identifying different pathotypes of plant pathogenic fungi.Particularly, the DNA sequences can be used as primers in PCR-basedanalysis for the identification of fungal pathotypes. The DNA sequencesof the invention include primers derived from partial sequences of themitochondrial small subunit ribosomal RNA genes (SSU rDNA) or theInternal Transcribed Spacer (ITS) sequences of the nuclear ribosomal RNAgene regions of particular fungal pathogens that are capable ofidentifying the particular pathogen.

[0035] Biomedical researchers have used PCR-based techniques for sometime and with moderate success to detect pathogens in infected animaltissues. Only recently, however, has this technique been applied todetect plant pathogens. The presence of Gaumannomyces graminis ininfected wheat has been detected using PCR of sequences specific to thepathogen mitochondrial genome (Schlesser et al., 1991, Applied andEnviron. Microbiol. 57: 553-556), and random amplified polymorphic DNA(i.e. RAPD) markers were able to distinguish numerous races ofGremmeniella abietina, the causal agent of scleroderris canker inconifers. U.S. Pat. No. 5,585,238 (herein incorporated by reference inits entirety) describes primers derived from the ITS sequences of theribosomal RNA gene region of strains of Septoria, Pseudocercosporella,and Mycosphaerella and their use in the identification of these fungalisolates using PCR-based techniques. In addition, U.S. Pat. No.5,955,274 (herein incorporated by reference in its entirety) describesprimers derived from the ITS sequences of the ribosomal RNA gene regionof strains of Fusarium and their use in the identification of thesefungal isolates using PCR-based techniques. Furthermore, U.S. Pat. No.5,800,997 (herein incorporated by reference in its entirety) describesprimers derived from the ITS sequences of the ribosomal RNA gene regionof strains of Cercospora, Helminthosporium, Kabatiella, and Puccinia andtheir use in the identification of these fungal isolates using PCR-basedtechniques.

[0036] Ribosomal genes are suitable for use as molecular probe targetsbecause of their high copy number. Despite the high conservation betweenmature rRNA sequences, the non-transcribed and transcribed spacersequences are usually poorly conserved and are thus suitable as targetsequences for the detection of recent evolutionary divergence. FungalrRNA genes are organized in units, each of which encodes three maturesubunits of 18S (small subunit), 5.8S, and 28S (large subunit). Thesesubunits are separated by two Internal Transcribed Spacers, ITS1 andITS2, of around 300 bp (White et al., 1990, in PCR Protocols, Innes etal., Eds., pages 315-322). In addition, the transcriptional units areseparated by non-transcribed spacer sequences (NTSs). ITS and NTSsequences are particularly suitable for the detection of specificpathotypes of different fungal pathogens.

[0037] Mitochondrial small subunit rDNA sequences similarly evolve morequickly than nuclear small subunit rDNA sequences and are thus moreuseful in differentiating more closely related species. As with the morequickly evolving ITS region sequences the mitochondrial small subunitrDNA sequences are composed of regions of higher and lesser variabilitywhich allow the use of conserved primers such as MS1 and MS2 describedby White et al. (1990, in PCR Protocols, Innes et al., Eds., pages315-322) to amplify out regions that contain more variability.

[0038] The DNA sequences of the invention are from partial sequences ofthe mitochondrial small subunit ribosomal RNA genes (SSU rDNA) or theInternal Transcribed Spacer sequences of the ribosomal RNA gene regionof different plant pathogens. The mitrochondrial SSU rDNA and nuclearITS region DNA sequences from different pathotypes within a pathogenspecies or genus vary among the different members of the species orgenus. Once the sequences of either of these regions has been determinedfor a given pathogen, these sequences can be aligned with otherrespective sequences from the same region for other pathogens. In thismanner, primers can be derived from the mitrochondrial SSU rDNA ornuclear ITS region sequences that are specific for a given pathogen.That is, primers can be designed based on regions within either themitrochondrial SSU or nuclear ITS region sequences that contain thegreatest differences in sequence among the fungal pathotypes whensimilar regions are compared. These sequences and primers based on thesesequences can be used to identify specific pathogens.

[0039] The present invention provides oligonucleotide primers for use inamplification-based detection of a fungal Internal Transcribed SpacerDNA sequence, wherein said primer has sequence identity with at least 10contiguous nucleotides of the Internal Transcribed Spacer sequence fromFusarium spp., such as but not limited to F. subglutinans, F.proliferatum, or F. verticillioides. In a preferred embodiment, thefungal specis is Fusarium proliferatum. In other preferred embodiments,the ITS comprises the nucleotides sequence of SEQ ID NO:5, 6, 7 or 8,more preferably, SEQ ID NO:7.

[0040] In preferred embodiments, oligonucleotide primers derived fromITS sequences comprises or consists of a nucleotide sequence of SEQ IDNOs: 9-12, 21 or 22. The primers are useful in the PCR-basedidentification of Fusarium proliferatum.

[0041] The present invention also provides oligonucleotide primers foruse in amplification-based detection of a fungal mitochondrial smallsubunit rDNA sequence, wherein said primer has sequence identity with atleast 10 contiguous nucleotides of the mitochondrial small subunitribosomal DNA sequence from Fusarium spp., in particular but not limitedto, F. subglutinans, F. verticillioides, or F. proliferatum. Moreparticularly, the mtSSU rDNA comprises the nucleotides sequence of SEQID NOs: 1-4.

[0042] In preferred embodiments, oligonucleotide primers derived frommitochondrial SSU rDNA comprise a nucleotide sequence of SEQ ID NOs:13-20, 23, or 24. The primers are useful in the PCR-based identificationof the Fusarium spp. pathogens of interest. In particular, the Fusariumspp. include, but are not limited to, F. subglutinans or F.verticillioides (syn. F. moniliforme). The present invention alsoprovides for pairs of oligonucleotide primers. In one embodiment, a pairof oligonucleotide primers for use in the amplification-based detectionof a fungal Internal Transcribed Spacer DNA sequence, wherein at leastone of said primers is the oligonucleotide primer has sequence identitywith at least 10 contiguous nucleotides of the Internal TranscribedSpacer sequence from Fusarium spp. such as but not limited to SEQ ID NO:5, 6, 7 or 8. In another embodiment, the invention provides a pair ofoligonucleotide primers, wherein at least one of said primers is theoligonucleotide primer of with at least 10 contiguous nucleotides of theInternal Transcribed Spacer sequence from a Fusarium proliferatum, suchas but not limited to SEQ ID NO:7.

[0043] In a preferred embodiment, the invention provides a pair ofoligonucleotide primers wherein at least one primer consists of thenucleotide sequence of SEQ ID NOS:9-12, 21 or 22. Preferred pairs ofprimers are: ITS1 (SEQ ID NO:9) and FPRO2 (SEQ ID NO:21); ITS1 (SEQ IDNO:9) and FPRO3 (SEQ ID NO:22); ITS3 (SEQ ID NO: 11) and FPRO2 (SEQ IDNO:21); and ITS3 (SEQ ID NO:I 1) and FPRO3 (SEQ ID NO:22).

[0044] In another embodiment, a pair of oligonucleotide primers for usein the amplification-based detection of a fungal mitochondrial smallsubunit ribosomal DNA sequence, wherein at least one of said primers isthe oligonucleotide primer has sequence identity with at least 10contiguous nucleotides of the mitochondrial small subunit ribosomal DNAsequence from Fusarium spp., such as but not limited to SEQ ID NOS: 14.In another embodiment, the invention provides a pair of oligonucleotideprimers, wherein at least one of said primers is the oligonucleotideprimer of with at least 10 contiguous nucleotides of the mitochondrialsmall subunit ribosomal DNA sequence from a Fusarium spp., such as butnot limited to SEQ ID NOS:1-4. In particular, the Fusarium spp. are butare not limited to, Fusarium subglutinans, Fusarium proliferatum and/orFusarium verticillioides (syn. F. moniliforme).

[0045] In a preferred embodiment, the a pair of oligonucleotide primerswherein one primer consists of a mitochondrial small subunit ribosomalDNA derived oligonucleotide primer of SEQ ID NOS: 13-20, 23, or 24.

[0046] In other more preferred embodiments, the invention provides pairsof oligonucleotide primers wherein said pair consists of SEQ ID NO: 15and SEQ ID NO: 16; wherein said pair consists of SEQ ID NO: 13 and SEQID NO: 16; wherein said pair consists of SEQ ID NO: 14 and SEQ ID NO:18; wherein said pair consists of SEQ ID NO: 14 and SEQ ID NO: 19; orwherein said pair consists of SEQ ID NO: 14 and SEQ ID NO:20.

[0047] Methods for the use of the primer sequences of the invention inPCR analysis are well known in the art. For example, see U.S. Pat. Nos.4,683,195 and 4,683,202, as well as Schlesser et al. (1991) Applied andEnviron. Microbiol. 57:553-556. See also, Nazar et al. (1991, Physiol.and Molec. Plant Pathol. 39:1 -11), which used PCR amplification toexploit differences in the ITS regions of Verticillium albo-atrum andVerticillium dahliae and therefore distinguish between the two species;and Johanson and Jeger (1993, Mycol. Res. 97: 670-674), who used similartechniques to distinguish the banana pathogens Mycosphaerella fjiensisand Mycosphaerella musicola.

[0048] The target DNA sequences of the invention can be cloned fromfungal pathogens by methods known in the art. In general, the methodsfor the isolation of DNA from fungal isolates are known. See, Raeder &Broda (1985) Letters in Applied Microbiology 2:17-20; Lee et al. (1990)Fungal Genetics Newsletter 35:23-24; and Lee and Taylor (1990) In: PCRProtocols: A Guide to Methods and Applications, Innes et al. (Eds.);pages 282-287.

[0049] The published mitochondrial SSU rDNA or ITS rDNA sequences arecompared within each pathogen group to locate divergences that might beuseful to test in PCR to distinguish the different species and/orstrains. From the identification of divergences, numerous primers aresynthesized and tested in PCR-amplification. Templates used forPCR-amplification testing are firstly purified pathogen DNA, andsubsequently DNA isolated from infected host plant tissue. Thus, it ispossible to identify pairs of primers that are diagnostic, i.e. thatidentified one particular pathogen species or strain but not anotherspecies or strain of the same pathogen. Primers are also designed toregions highly conserved among the species to develop genus-specificprimers as well as primers that will identify any of several fungalpathogens that cause a particular disease. For example, primers aredeveloped to differentiate species of Fusarium: F. proliferatum, F.verticillioides, and F. subglutinans.

[0050] Preferred primer combinations are able to distinguish between thedifferent species or strains in infected host tissue, i.e. host tissuethat has previously been infected with a specific pathogen species orstrain. This invention provides numerous primer combinations thatdistinguish Fusarium proliferatum, F. verticillioides, and F.subglutinans. The primers of the invention are designed based onsequence differences among either the mitochondrial SSU rDNA or the ITSrDNA regions. A minimum of one base pair difference between sequencescan permit design of a discriminatory primer. Primers designed to aspecific fungal DNA sequence can be used in combination with a primermade to a conserved sequence region flanking the region containingdivergences to amplify species-specific PCR fragments. In general,primers should have a theoretical melting temperature between about 60to about 70 degree ° C. to achieve good sensitivity and should be voidof significant secondary structure and 3′ overlaps between primercombinations. In preferred embodiments, primers are anywhere fromapproximately 5-30 nucleotide bases long.

[0051] In one embodiment, the present invention provides a method forthe detection of a fungal pathogen, comprising the steps of:

[0052] (a) isolating DNA from a plant tissue infected with a pathogen;

[0053] (b) subjecting said DNA to polymerase chain reactionamplification using at least one primer having sequence identity with atleast 10 contiguous nucleotides of an Internal Transcribed Spacersequence of a Fusarium spp.; and

[0054] (c) detecting said fungal pathogen by visualizing the product orproducts of said polymerase chain reaction amplification.

[0055] In preferred embodiments, the method detects infections with apathogen, wherein said fungal pathogen Fusarium subglutinans, Fusariumproliferatum or Fusarium verticillioides. In another preferredembodiment, the ITS sequences have the nucleotide sequence of SEQ IDNO:5, 6, 7, or 8.

[0056] In another preferred embodiment, the method uses at least oneprimer having the nucleotide sequence of SEQ ID NOS: 9-12, 20 or 21. Inanother embodiment, the present invention provides for a method for thedetection of a fungal pathogen, comprising the steps of:

[0057] (a) isolating DNA from a plant tissue infected with a pathogen;

[0058] (b) subjecting said DNA to polymerase chain reactionamplification using at least one primer having sequence identity with atleast 10 contiguous nucleotides of a mitochondrial small subunit rDNAsequence of a Fusarium spp. ; and

[0059] (c) detecting said fungal pathogen by visualizing the product orproducts of said polymerase chain reaction amplification.

[0060] In preferred embodiments, the method detects the fungal pathogensof Fusarium subglutinans, Fusarium proliferatum or Fusariumverticillioides.

[0061] In another preferred embodiment, the method uses at least oneprimer having the nucleotide sequence of SEQ ID NOS: 13-20, 23 or 24.

[0062] In more preferred embodiments, the methods uses a pairs ofoligonucleotide primers wherein said pair consists of SEQ ID NO: 15 andSEQ ID NO: 16; wherein said pair consists of SEQ ID NO:13 and SEQ IDNO:16; wherein said pair consists of SEQ ID NO:14 and SEQ ID NO:18;wherein said pair consists of SEQ ID NO:14 and SEQ ID NO:19; or whereinsaid pair consists of SEQ ID NO:14 and SEQ ID NO:20.

[0063] The present invention lends itself readily to the preparation of“kits” containing the elements necessary to carry out the process. Sucha kit may comprise a carrier being compartmentalized to receive in closeconfinement therein one or more container, such as tubes or vials. Oneof the containers may contain unlabeled or detectably labeled DNAprimers. The labeled DNA primers may be present in lyophilized form orin an appropriate buffer as necessary. One or more containers maycontain one or more enzymes or reagents to be utilized in PCR reactions.These enzymes may be present by themselves or in admixtures, inlyophilized form or in appropriate buffers.

[0064] In one embodiment, the diagnostic kit used in detecting a fungalpathogen, comprises at least one primer of SEQ ID NOs: 9-12, 21 or 22for ITS derived primers or SEQ ID NOs: 13-20, 23, or 24 for primersderived from mitochondrial small subunit ribosomal DNA.

[0065] In more preferred embodiments, the diagnostic kit used indetecting a fungal pathogen, comprises the pair of primers describedabove. More preferably, the pairs of primers are SEQ ID NO: 15 and SEQID NO: 16; SEQ ID NO: 13 and SEQ ID NO: 16; SEQ ID NO: 14 and SEQ ID NO:18; SEQ ID NO: 14 and SEQ ID NO: 19; or SEQ ID NO: 14 and SEQ ID NO:20.

[0066] Finally, the kit may contain all of the additional elementsnecessary to carry out the technique of the invention, such as buffers,extraction reagents, enzymes, pipettes, plates, nucleic acids,nucleoside triphosphates, filter paper, gel materials, transfermaterials, autoradiography supplies, and the like.

[0067] The examples below show typical experimental protocols that canbe used in the selection of suitable primer sequences, the testing ofprimers for selective and diagnostic efficacy, and the use of suchprimers for disease and fungal isolate detection. Such examples areprovided by way of illustration and not by way of limitation.

[0068] Numerous references cited above are all incorporated herein intheir entireties.

EXAMPLES

[0069] Standard recombinant DNA and molecular cloning techniques usedhere are well known in the art and are described by J. Sambrook, E. F.Fritsch and T. Maniatis, Molecular Cloning: A Laboratory manual, ColdSpring Harbor laboratory, Cold Spring Harbor, N.Y. (1989) and by T. J.Silhavy, M. L. Berman, and L. W. Enquist, Experiments with Gene Fusions,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and byAusubel, F. M. et al., Current Protocols in Molecular Biology, pub. byGreene Publishing Assoc. and Wiley-Interscience (1987).

Example 1 Fungal Isolates and Genomic Fungal DNA Extraction

[0070] See Tables 1 and 2 for listings of the fungal isolates used andtheir sources. Isolates used to validate the assays in the followingexamples were obtained from a number of academic institutions andcollections (Table 1). TABLE 1 Source of Test Isolates Isolate SourceIsolation Geographic Origin Fusarium moniliforme M-1231 Penn State¹ RicePhilippines Fusarium moniliforme M-1264 Penn State¹ Rice Sierra LeoneFusarium moniliforme M-1329 Penn State¹ Rice California, USA Fusariummoniliforme M-3120 Penn State¹ Maize California, USA Fusariummoniliforme M-3125 Penn State¹ Maize California, USA Fusariumsporotrichioides 3299 NRRL² Fusarium subglutinans M-3693 Penn State¹Maize Iowa, USA Fusarium subglutinans M-3696 Penn State¹ Maize Iowa, USAFusarium moniliforme M-3744 Penn State¹ Rice Australia Fusariummoniliforme M-5167 Penn State¹ Rice Iran Fusarium moniliforme M-5587Penn State¹ Date Palm Iraq Fusarium moniliforme M-5605 Penn State¹Poland Fusarium proliferatum M-5991 Penn State¹ Swine Feed Iowa, USAFusarium moniliforme M-6173 Penn State¹ Rice Malaysia Fusariumsambucinum- R-6380 Penn State¹ Potato Germany sulphureum Fusariummoniliforme M-6471 Penn State¹ Maize Kansas Fusarium moniliforme M-8510Penn State¹ Rice Nepal Fusarium moniliforme 6396 NRRL² Chicken Arkansas,USA Feed Fusarium moniliforme 13563 NRRL² Pinus taeda North Carolina,USA Fusarium moniliforme 25029 NRRL² Nilaparvata India lugens Fusariumsubglutinans 13588 NRRL² Maize Iowa, USA Fusarium subglutinans 13599NRRL² Maize Zambia Fusarium subglutinans 20844 NRRL² Maize GermanyFusarium proliferatum 94-041 Iowa State³ Maize Iowa Fusariumproliferatum 94-066 Iowa State³ Maize Iowa Fusarium proliferatum 94-129Iowa State³ Maize Iowa Fusarium proliferatum 95-122 Iowa State³ MaizeIowa Fusarium proliferatum 95-135 Iowa State³ Maize Iowa Fusariumproliferatum 95-289 Iowa State³ Maize Iowa Fusarium culmorum R-5126 PennState¹ Minnesota, USA Fusarium graminearum R-8637 Penn State¹ Settat,Morocco Microdochium nivale 15N1 S. Edwards⁴ United Kingdom M. nivalevar. majus 93 Novartis, Basel⁵ — Fusarium poae T-427 Penn State¹Pennsylvannia, USA Fusarium avenaceum 64452 ATCC⁶ Wheat Poland Diplodiamaydis 5139 C. Naidoo⁷ Illinois, USA Macrophomina phaseolina MP97 J.Mihail⁸ Missouri, USA Aspergillus flavus 3557 NRRL Collection²Kabatiella zeae 18594 ATCC⁶ Maize Wisconsin, USA Cercospora zeae-maydis69281L C. Naidoo⁷ Illinois, USA Cercospora zeae-maydis 26158 ATCC⁶ MaizeNew York, USA Puccinia sorghi VA Helminthosporium maydis 24772 ATCC⁶Maize North Carolina, USA Helminthosporium maydis 11534 ATCC⁶ MaizeMaryland, USA Helminthosporium 16185 ATCC⁶ Maize Virginia, USA carbonumHelminthosporium 24962 ATCC⁶ Maize Illinois, USA carbonumHelminthosporium turcicum 26306 ATCC⁶ Maize Illinois, USA Fusariumculmorum 62215 ATCC⁶ Wheat seed Switzerland Fusarium culmorum R-5106Darling Downs, Australia

[0071] Unknown ear rot isolates cultured from field grown maize wereobtained from the Novartis Seeds research station in Stanton, Minn., USAand are described in Table 2. Fungi are grown on PDA (Potato DextroseAgar) plates. Cultures are incubated for up to 10 days at 28° C. Myceliaare ground in liquid nitrogen, and total genomic DNA is extracted usingthe protocol of Lee and Taylor (1990; In: PCR Protocols: A Guide toMethods and Applications; Eds.: Innes et al.; pages 282-287). TABLE 2Geographical Source of Unknown Ear Rot Isolates Isolate GeographicalDesignation Region Fm001 Nebraska Fm002 Georgia Fm003 Iowa Fm004 OhioFm005 Illinois Fm006 Illinois Fm007 Illinois Fm008 Illinois Fm009 OhioFm010 Ohio Fm011 Fm012 Ohio Fm013 Kentucky Fm014 Illinois Fm034 KentuckyFm035 Illinois Fm036 Fm037 Fm039 Hawaii Fm040 Hawaii Fm041 NorthCarolina Fm042 North Carolina Fm043 Colorado Fm044 Mississippi Fm045Hawaii Fm046 Hawaii Fm047 Hawaii Fm048 Hawaii Fm049 Hawaii Fm050 HawaiiFm051 Hawaii Fm052 Hawaii Fm053 Hawaii Fm054 Hawaii Fm055 Hawaii Fm056Hawaii Fsub1 Minnesota Fsub2 Minnesota Fsub3 Minnesota Fsub4 MinnesotaBC3 189 Minnesota

Example 2 DNA Extraction from Maize Tissues

[0072] DNA is extracted from maize tissues by one of two methods. Themethod described in Example 2A is used for bulk extractions of maizeleaves taken from some 10 -15 plants at either the ear, the node abovethe ear, or the node below the ear. Example 2B describes a method usedfor extracting DNA from maize tissues in 1.5 mL tubes. This method maybe used for concentrating the sample around one lesion or for testinganther or axil material.

Example 2A Large-Scale DNA Extraction from Maize Leaves

[0073] DNA is extracted from maize leaves in a bulk maceration asfollows:

[0074] (1) A sample consists of whole maize leaves collected from some20 plants from the same position on the plant (ear leaf, third ear belowleaf, etc.) and kept separated accordingly. The top third of each leafis taken and extracted in bulk.

[0075] (2) The sample is placed in a Bioreba (Reinach, Switzerland)heavy duty plastic bag (cat#490100). The plant tissue is weighed,plastic bag with leaves minus the tare (weight of the plastic bag).

[0076] (3) An equal volume (ml) of CTAB Extraction Buffer (100 mM Tris,pH 8.0; 1.4 M NaCl; 20 mM Na₂-EDTA; 2% Hexadecyltrimethyl ammoniumbromide (CTAB); 2 % Polyvinylpyrolidine (PVPP); 0.1% ascorbic acid; 0.2%β-mercaptoethanol) is added perweight (g) of maize tissue. The tissue ismacerated using a Bioreba Homex 6 homogenizer set at 70. The tissue isground until fibrous.

[0077] (4) The extraction juice is homogenized and is aliquoted intoeppendorf tubes on ice.

[0078] (a) The concentrated extract is boiled for 5 minutes.

[0079] (b) The boiled extract is placed on ice for two minutes. Theboiled extract 5 is then centrifuged for 5 minutes at 10,000×G.

[0080] (c) 1:40 dilutions of the supernatant from the microfuged extractin cold dH₂0 are made and used as sample DNA template in PCR assays.

[0081] (d) The diluted extracts are stored on ice until ready to use.

[0082] For the purpose of showing that the assays do not cross-reactwith maize tissue, a 1o sample of field-grown maize visually assessed ashealthy obtained from Franklin, Id., USA near the end of June 1999 isused to test for background effects. DNA preparations are made from thesample using the protocol outlined in this example (The extract isdesignated 1999 Maize sample #1).

Example 2B Small-Scale DNA Extraction From Anther, Axil, and HuskTissues Collected from Field-Grown Maize.

[0083] Samples of Maize tissues consisting of anther, axil, or huskmaterial are received in eppendorf tubes. Sample sizes are limited tooccupying ⅕ volume of the 1.5 mL tube:

[0084] (1) Check/set the temperature of the dry bath is at 90° C.Transport samples on Dry-ice to Sawz-all. Keep samples on Dry-ice or atminus 80° C. before and after grinding.

[0085] (2) Place samples in box with lid to fit in a high velocityshaking apparatus.

[0086] (3) Secure the box in the shaking apparatus with extra lid andcardboard to ensure a tight fit. Grind for one minute. Remove box.Rotate 180° and grind for an additional 25 minute.

[0087] (4) Add 500 μL of extraction buffer (100 mM Tris 8.0, 10 mM EDTA,1% Sarkosyl)

[0088] (5) Vortex tubes

[0089] (6) Place tubes in a 90° C. dry bath. Incubate samples for 30minutes.

[0090] (7) Remove tubes from bath and cool on ice >5 minutes.

[0091] (8) Centrifuge sample at 10,000 rpm for 5 minutes at roomtemperature.

[0092] (9) 1 μL of a 1:20 dilution of the supernatant serves as templatefor PCR. Diluted samples should be stored at minus 20° C. and kept onice for all manipulations.

[0093] Maize tissue samples extracted by the above method and used inthe following Examples are listed in Table 3. TABLE 3 Maize TissueSamples¹ Sample Designation Tissue H-5 Husk H-9 Husk SBP-2 Huskassociated with Sap Beetle

Example 3 Polymerase Chain Reaction (PCR) Amplification

[0094] Polymerase chain reactions are performed with the GeneAmp Kitfrom Perkin-Elmer (Foster City, Calif.; part no. N808-0009) using 50 mMKCl, 2.5 mM MgCl₂, 10 mM Tris-HCl, pH8.3, containing 200 μM of eachdTTP, DATP, dCTP, and dGTP in 25 μL reactions containing 25 pmol eachprimer, 1.25 units of Taq polymerase and 10 ng of genomic DNA. Reactionsare run for 30-40 cycles of 15 s at 94° C., 15 s at 50° C.-70° C., and45 s at 72° C. in a Perkin-Elmer Model 9600 or 9700 thermal cycler. Theproducts are analyzed by loading 10 μl of each PCR sample on a 1.0%agarose gel and electrophoresing.

Example 4 Synthesis and Purification of OligonucleotidesOligonucleotides (Primers) are Synthesized by, for Example, eitherIntegrated DNA Technologies (Coralville, Iowa) or Midland CertifiedReagent Company (Midland, Tex.). Example 5 Design of Species-SpecificPCR Primers

[0095] Sequences are obtained from the GenBank database of the NationalCenter for Biotechnology Information (www.ncbi.nlm.nih.gov) for partialsequence listings of small subunit ribosomal RNA and mitochondrial genefor F. verticillioides (SEQ ID NO: 1); F. proliferatum (SEQ ID NO:2); F.graminearum (syn. Gibberella zeae) (SEQ ID NO:3); and F. subglutinans(SEQ ID NO:4). A multiple sequence alignment is made of these sequences.The alignment is analyzed for divergences among the four sequences. Thedivergences permit the development of primers that will specificallyamplify one of the four target sequences in PCR reactions.Oligonucleotide primers are designed to target regions that contain thegreatest differences in sequence among the species analyzed (Table 4).FSUB1 (SEQ ID NO: 15), FSUB2 (SEQ ID NO: 16), and FSUB3 (SEQ ID NO: 17)are designed to target the mitochondrial small subunit (mtSSU) rDNA ofFusarium subglutinans. FPRO1 (SEQ ID NO:20) is designed to target themtSSU rDNA of Fusarium proliferatum. The mtSSU rDNA of Fusariumverticillioides is the target of primers FVERT1 (SEQ ID NO: 18) andFVERT2 (SEQ ID NO: 19). These primers may be used in combination withprimers FCORN1 (SEQ ID NO: 13) and FCORN2 (SEQ ID NO: 14) that targetmtSSU rDNA conserved between the three targeted species of Fusarium.

[0096] Similarly, ITS region rDNA sequence listings for F. subglutinans(SEQ ID NO:5), F. graminearum (syn. Gibberella zeae) (SEQ ID NO:6), F.proliferatum (SEQ ID NO:7), and F. verticillioides (syn. F.verticillioides) (SEQ ID NO:8) were obtained. An alignment of ITS regionsequences is used as above to develop specific primers. In addition, thepublished ribosomal gene-specific primers ITS1, ITS2, ITS3 and ITS4(White et al., 1990; In: PCR Protocols; Eds.: Innes et al. pages315-322) are synthesized for testing in combination with the primersspecific for the ITS regions. Primers FPRO2 and FPRO3 target the nuclearrDNA ITS 2 region of Fusarium proliferatum. They may be used with ITS1,the conserved fungal nuclear rDNA primer targeting the ITS1 region. Thespecies-specific primers as well as the conserved fungal ITS regionprimers are shown in Table 4. TABLE 4 Primers Designed for Detection ofFusarium Ear Rot Pathogens Fusarium subglutinans, F. proliferatum, andF. verticillioides Name Oligo Sequence (5′→3′) Target Identifier ITS1TCCGTAGGTGAACCTGCGG Fungal Nuclear rDNA ITS region SEQ-ID-NO:9 ITS2GCTGCGTTCTTCATCGATGC Fungal Nuclear rDNA ITS region SEQ-ID-NO:10 ITS3GCATCGATGAAGAACGCAGC Fungal Nuclear rDNA ITS region SEQ-ID-NO:11 ITS4TCCTCCGCTTATTGATATGG Fungal Nuclear rDNA ITS region SEQ-ID-NO:12 FCORN1GCAACTTGGAGAAGTGGCAAG Fusarium sp. Mitochondrial SEQ-ID-NO:13 smallsubunit rDNA FCORN2 AAGTCTTCCAGTATGGGGAG Fusarium sp. MitochondrialSEQ-ID-NO:14 small subunit rDNA FSUB1 GTGCGATATCTTTAGGAGGC Fusariumsubglutinans SEQ-ID-NO:15 Mitochondrial small subunit rDNA FSUB2TGAACTAGACTACCAACTCAG Fusarium subglutinans SEQ-ID-NO:16 Mitochondrialsmall subunit rDNA FSUB3 CAAATCTAAGGCTGGCTTGTA Fusarium subglutinansSEQ-ID-NO:17 Mitochondrial small subunit rDNA FVERT1TGGTGGACTAGTCTGAATCC Fusarium verticillioides SEQ-ID-NO:18 Mitochondrialsmall subunit rDNA FVERT2 TGAACTACGAGTAACCCACC Fusarium verticillioidesSEQ-ID-NO:19 Mitochondrial small subunit rDNA FPRO1TAAACTAACTCAACTAGACGAG Fusarium proliferatum SEQ-ID-NO:20 Mitochondrialsmall subunit rDNA FPRO2 GATTTCGGGGCCGGCTTGC Fusarium proliferatumnuclear SEQ-ID-NO:21 rDNA ITS region FPRO3 CGCAAGGGCTCGCCGATC Fusariumproliferatum nuclear SEQ-ID-NO:22 rDNA ITS region MS1CAGCAGTCAAGAATATTAGTCAATG Fungal mitochondrial small subunitSEQ-ID-NO:23 rDNA region MS2 GCGGATTATCGAATTAAATAAC Fungal mitochondrialsmall subunit SEQ-ID-NO:24 rDNA region

Example 6 Determination of Primer Specificity to Purified Fungal GenomicDNA

[0097] PCRs are performed according to Example 3 using different primercombinations (Table 5) in an attempt to amplify single specificfragments. Specific PCR amplification products are produced from primersdesigned from the mitochondrial small subunit rDNA or the nuclear rDNAITS regions of each fungal strain of interest.

[0098] In an initial screen for specificity, PCR reaction mixtures aremade according to Example 3 for each of the primer combinations in Table5. These are run against a negative control (no DNA added), a healthymaize tissue control (prepared in Example 2A) to test for backgroundamplification, and 10 ng of DNA from the following isolates in Table 1:Fusarium moniliforme M-3120; Fusarium subglutinans M-3693; Fusariumsubglutinans M-3696; Fusarium proliferatum M-5991; Fusarium culmorumR-5126; Fusarium graminearum R-8637; Microdochium nivale 15N1; M. nivalevar. majus 93; Fusarium poae T427; and Fusarium avenaceum 64452 preparedas described in Example 1. TABLE 5 Possible Combinations of PCR Primersfor the Specific Amplification of Fusarium subglutinans, F.verticillioides, and F. proliferatum. Target Approximate Pathogen 5′primer 3′ primer Product Size (bp) Fusarium subglutinans FCORN1 (SEQ IDNO: 13) FSUB2 (SEQ ID NO: 16) 513 Fusarium subglutinans FCORN2 (SEQ IDNO: 14) FSUB2 (SEQ ID NO: 16)  495¹ Fusarium subglutinans FSUB1 (SEQ IDNO: 15) FSUB2 (SEQ ID NO: 16) 456 Fusarium subglutinans FCORN1 (SEQ IDNO: 13) FSUB3 (SEQ ID NO: 17)  559² Fusarium subglutinans FCORN2 (SEQ IDNO: 14) FSUB3 (SEQ ID NO: 17)  541³ Fusarium subglutinans FSUB1 (SEQ IDNO: 15) FSUB3 (SEQ ID NO: 17)  502⁴ Fusarium verticillioides FCORN1 (SEQID NO: 13) FVERT1 (SEQ ID NO: 18)  544⁵ Fusarium verticillioides FCORN2(SEQ ID NO: 14) FVERT1 (SEQ ID NO: 18) 526 Fusarium verticillioidesFCORN1 (SEQ ID NO: 13) FVERT2 (SEQ ID NO: 19)  505⁶ Fusariumverticillioides FCORN2 (SEQ ID NO: 14) FVERT2 (SEQ ID NO: 19) 487Fusarium proliferatum FCORN1 (SEQ ID NO: 13) FPRO1 (SEQ ID NO: 20)  520⁷Fusarium proliferatum FCORN2 (SEQ ID NO: 14) FPRO1 (SEQ ID NO: 20) 502Fusarium proliferatum ITS1 (SEQ ID NO: 9) FPRO2 (SEQ ID NO: 21)  385⁸Fusarium proliferatum ITS1 (SEQ ID NO: 9) FPRO3 (SEQ ID NO: 22)  370⁹Fusarium proliferatum ITS3 (SEQ ID NO: 11) FPRO2 (SEQ ID NO: 21) 180Fusarium proliferatum ITS3 (SEQ ID NO: 11) FPRO3 (SEQ ID NO: 22) 160Fungal ITS region ITS1 (SEQ ID NO: 9) ITS4 (SEQ ID NO: 12) 530 FungalITS region ITS1 (SEQ ID NO: 9) ITS2 (SEQ ID NO: 10) 210 Fungal ITSregion 1TS3 (SEQ ID NO: 9) ITS4 (SEQ ID NO: 12) 330

[0099] When visualized on an ethidium bromide stained gel, severalprimer pairs amplified single products from target DNA with all otherreactions (negative control, maize background, and other fungal DNAs)free of both specific and nonspecific reaction products. The primerpairs that give the best amplification for their specific targets withno cross-amplification are summarized in Table 6. See footnotes (Table5) for information on those primer pairs that amplified target DNA butwith less satisfactory results in terms of specificity. TABLE 6 PCRPrimer Pairs Providing Specific and Sensitive Amplification of TargetDNA for Fusarium subglutinans, F. verticillioides, and F. proliferatumPCR Assays. Target Approximate Pathogen 5′ primer 3′ primer Product Size(bp) Fusarium subglutinans FSUB 1 (SEQ ID NO: 15) FSUB2 (SEQ ID NO: 16)456 Fusarium subglutinans FCORN1 (SEQ ID NO: 13) FSUB2 (SEQ ID NO: 16)513 Fusarium verticillioides FCORN2 (SEQ ID NO: 14) FVERT1 (SEQ ID NO:18) 526 Fusarium verticillioides FCORN2 (SEQ ID NO: 14) FVERT2 (SEQ IDNO: 19) 487 Fusarium proliferatum FCORN2 (SEQ ID NO: 14) FPRO1 (SEQ IDNO: 20) 502

Example 7 Validation of Fusarium subglutinans, F. verticillioides, andF. proliferatum

[0100] PCR Assays Showing Reactivity of Multiple Isolates for a GivenTarget.

[0101] One of the primer pairs in Table 6 is chosen for each target DNAfor further characterization and testing: FSUB1 and FSUB2 for Fusariumsubglutinans, FCORN2 and FVERT1 for F. verticillioides, and FCORN2 withFPRO1 for F. proliferatum. Each is run in PCR mastermixes against DNAsfrom a panel of fungal species (all isolates in Table 1) prepared as inExample 1. Products are visualized on an ethidium bromide stained gel.Results are scored as either positive (+) or negative (−) for theamplification of target DNA with any product visible, of the correctsize, being considered a positive and with nonspecifics recorded ifpresent. Results of each of these tests are shown in Tables 7-9. Table 7shows that primers FSUB1 (SEQ ID NO:15) and FSUB2 (SEQ ID NO:16), whenprepared in PCR reactions as described in Example 3, amplify target DNAfrom only the isolates identified as Fusarium subglutinans. The primersdo not react with isolates of Fusarium proliferatum, F. verticillioides,or with other fungal species known to infect or colonize maize tissue.This experiment also shows that the F. subglutinans specific primers donot react with a preparation of maize DNA described in Example 2A. TABLE7 Results of F. subglutinans PCR Assay Against a Panel of Ear RotPathogen DNAs and a Maize Background Check. F. subglutinans Fungalspecies Isolate Isolation Geographic Origin PCR Result Fusariumproliferatum M-5991 Swine Feed Iowa, USA − Fusarium proliferatum 94-041Maize Iowa, USA − Fusarium proliferatum 94-066 Maize Iowa, USA −Fusarium proliferatum 94-129 Maize Iowa, USA − Fusarium proliferatum95-122 Maize Iowa, USA − Fusarium proliferatum 95-135 Maize Iowa, USA −Fusarium proliferatum 95-289 Maize Iowa, USA − Fusarium proliferatumM-1231 Rice Phillipines − Fusarium proliferatum M-1264 Rice Sierra Leone− Fusarium proliferatum M-1329 Rice California, USA − Fusariumproliferatum M-3744 Rice Australia − Fusarium proliferatum M-5167 RiceIran − Fusarium proliferatum M-5587 Date Palm Iraq − Fusariumproliferatum M-5605 Poland − Fusarium proliferatum M-6173 Rice Malaysia− Fusarium proliferatum M-6471 Maize Kansas, USA − Fusarium proliferatumM-8510 Rice Nepal, USA − Fusarium verticillioides NRRL Chicken Arkansas,USA − 6396 Feed Fusarium verticillioides NRRL Pinus taeda NorthCarolina, − 13563 USA Fusarium verticillioides M-3120 Maize California,USA − Fusarium verticillioides M-3125 Maize California, USA − Fusariumsubglutinans NRRL Maize Iowa, USA + 13588 Fusarium subglutinans NRRLMaize Zambia + 13599 Fusarium subglutinans NRRL Maize Germany + 20844Fusarium subglutinans M3693 Maize Iowa, USA + Fusarium subglutinansM3696 Maize Iowa, USA + Fusarium sambucinium- R-6380 Maize Iowa, USA −sulphureum Fusarium 3299 − sporotrichioides Fusarium culmorum R-5126Minnesota, USA − Fusarium graminearum R-8637 Settat, Morocco −Microdochium nivale 15N1 United Kingdom − Microdochium nivale #093 −var. majus Fusarium poae T-427 Pennsylvannia, − USA Fusarium avenaceumATCC Poland − 64452 Diplodia maydis 5139 Macrophomina MP97 phaseolinaAspergillus flavus 3557 Kabatiella zeae 18594 Maize Wisconsin, USA −Cercospora zeae-maydis 6928IL Cercospora zeae-maydis 26158 Maize NewYork, USA − Puccinia sorghi VA − Helminthosporium 24772 Maize NorthCarolina, − maydis USA Helminthosporium 11534 Maize Maryland, USA −maydis Helminthosporium 16185 Maize Virginia, USA − carbonumHelminthosporium 24962 Maize Illinois, USA − carbonum Helminthosporium26306 Maize Illinois, USA − turcicum Fusarium culmorum 62215 Wheat seedSwitzerland − Fusarium culmorum R-5106 Darling Downs, − Australia 1999Maize sample #1 — — Iowa, USA −

[0102] Table 8 shows that primers FCORN2 (SEQ ID NO: 14) and FPRO1 (SEQID NO:20), when prepared in PCR reactions as described in Example 3,amplify target DNA from only the isolates identified as Fusariumproliferatum and with all isolates in this study that were identified asF. proliferatum. The primers do not react with maize DNA (1999 Maizesample #1) or with other fungal species known to infect or colonizemaize tissue including F. verticillioides and F. subglutinans. TABLE 8Results of F. proliferatum PCR Assay Against a Panel of Ear Rot PathogenDNAs and a Maize Background Check. F. proliferatum Fungal speciesIsolate Isolation Geographic Origin PCR Result Fusarium proliferatumM-5991 Swine Feed Iowa, USA + Fusarium proliferatum 94-041 Maize Iowa,USA + Fusarium proliferatum 94-066 Maize Iowa, USA + Fusariumproliferatum 94-129 Maize Iowa, USA + Fusarium proliferatum 95-122 MaizeIowa, USA + Fusarium proliferatum 95-135 Maize Iowa, USA + Fusariumproliferatum 95-289 Maize Iowa, USA + Fusarium proliferatum M-1231 RicePhillipines + Fusarium proliferatum M-1264 Rice Sierra Leone + Fusariumproliferatum M-1329 Rice California, USA + Fusarium proliferatum M-3744Rice Australia + Fusarium proliferatum M-5167 Rice Iran + Fusariumproliferatum M-5587 Date Palm Iraq + Fusarium proliferatum M-5605Poland + Fusarium proliferatum M-6173 Rice Malaysia + Fusariumproliferatum M-6471 Maize Kansas, USA + Fusarium proliferatum M-8510Rice Nepal, USA + Fusarium verticillioides NRRL Chicken Arkansas, USA −6396 Feed Fusarium verticillioides NRRL Pinus taeda North Carolina, −13563 USA Fusarium verticillioides M-3120 Maize California, USA −Fusarium verticillioides M-3125 Maize California, USA − Fusariumsubglutinans NRRL Maize Iowa, USA − 13588 Fusarium subglutinans NRRLMaize Zambia − 13599 Fusarium subglutinans NRRL Maize Germany − 20844Fusarium subglutinans M3693 Maize Iowa, USA − Fusarium subglutinansM3696 Maize Iowa, USA − Fusarium sambucinium- R-6380 Maize Iowa, USA −sulphureum Fusarium 3299 sporotrichioides Fusarium culmorum R-5126Minnesota, USA − Fusarium graminearum R-8637 Settat, Morocco −Microdochium nivale 15N1 United Kingdom − Microdochium nivale #093 var.majus Fusarium poae T-427 Pennsylvannia, − USA Fusarium avenaceum ATCCPoland − 64452 Diplodia maydis 5139 Macrophomina MP97 phaseolinaAspergillus flavus 3557 Kabatiella zeae 18594 Maize Wisconsin, USA −Cercospora zeae-maydis 6928IL Cercospora zeae-maydis 26158 Maize NewYork, USA − Puccinia sorghi VA Helminthosporium 24772 Maize NorthCarolina, − maydis USA Helminthosporium 11534 Maize Maryland, USA −maydis Helminthosporium 16185 Maize Virginia, USA − carbonumHelminthosporium 24962 Maize Illinois, USA − carbonum Helminthosporium26306 Maize Illinois, USA − turcicum Fusarium culmorum 62215 Wheat seedSwitzerland − Fusarium culmorum R-5106 Darling Downs, − Australia 1999Maize sample #1 — — Iowa, USA −

[0103] The primers FCORN2 (SEQ ID NO: 14) and FVERT1 (SEQ ID NO: 18)were run against the same DNA preparations of fungal isolates and maizetissue that were tested using the F. subglutinans and F. proliferatumspecific primers (results in Tables 7 and 8, respectively). The F.verticillioides specific primers, when prepared in PCR reactions asdescribed in Example 3, amplify target DNA from only the isolatesidentified as Fusarium verticillioides (Table 9). The primers do notreact with isolates of Fusarium subglutinans, F. proliferatum, or withother fungal species known to infect or colonize maize tissue. Table 9also shows that FCORN2 and FVERT1 do not react with a preparation ofmaize DNA. TABLE 9 Results of F. verticillioides PCR Assay Against aPanel of Ear Rot Pathogen DNAs and a Maize Background Check. GeographicF. verticillioides Fungal species Isolate Isolation Origin PCR ResultFusarium proliferatum M-5991 Swine Feed Iowa, USA − Fusariumproliferatum 94-041 Maize Iowa, USA − Fusarium proliferatum 94-066 MaizeIowa, USA − Fusarium proliferatum 94-129 Maize Iowa, USA − Fusariumproliferatum 95-122 Maize Iowa, USA − Fusarium proliferatum 95-135 MaizeIowa, USA − Fusarium proliferatum 95-289 Maize Iowa, USA − Fusariumproliferatum M-1231 Rice Phillipines − Fusarium proliferatum M-1264 RiceSierra Leone − Fusarium proliferatum M-1329 Rice California, USA −Fusarium proliferatum M-3744 Rice Australia − Fusarium proliferatumM-5167 Rice Iran − Fusarium proliferatum M-5587 Date Palm Iraq −Fusarium proliferatum M-5605 Poland − Fusarium proliferatum M-6173 RiceMalaysia − Fusarium proliferatum M-6471 Maize Kansas, USA − Fusariumproliferatum M-8510 Rice Nepal, USA − Fusarium verticillioides NRRLChicken Arkansas, USA + 6396 Feed Fusarium verticillioides NRRL Pinustaeda North Carolina, + 13563 USA Fusarium verticillioides M-3120 MaizeCalifornia, USA + Fusarium verticillioides M-3125 Maize California,USA + Fusarium subglutinans NRRL Maize Iowa, USA − 13588 Fusariumsubglutinans NRRL Maize Zambia − 13599 Fusarium subglutinans NRRL MaizeGermany − 20844 Fusarium subglutinans M3693 Maize Iowa, USA − Fusariumsubglutinans M3696 Maize Iowa, USA − Fusarium sambucinium- R-6380 MaizeIowa, USA − sulphureum Fusarium sporotrichioides 3299 − Fusariumculmorum R-5126 Minnesota, USA − Fusarium graminearum R-8637 Settat,Morocco − Microdochium nivale 15N1 United Kingdom − Microdochium nivale#093 − var. majus Fusarium poae T-427 Pennsylvannia, − USA Fusariumavenaceum ATCC Poland − 64452 Diplodia maydis 5139 − Macrophominaphaseolina MP97 − Aspergillus flavus 3557 − Kabatiella zeae 18594 MaizeWisconsin, USA − Cercospora zeae-maydis 6928IL − Cercospora zeae-maydis26158 Maize New York, USA − Puccinia sorghi VA − Helminthosporium maydis24772 Maize North Carolina, − USA Helminthosporium maydis 11534 MaizeMaryland, USA − Helminthosporium 16185 Maize Virginia, USA − carbonumHelminthosporium 24962 Maize Illinois, USA − carbonum Helminthosporium26306 Maize Illinois, USA − turcicum Fusarium culmorum 62215 Wheat seedSwitzerland − Fusarium culmorum R-5106 Darling Downs, − Australia 1999Maize sample #1 — — Iowa, USA −

[0104] In summary, assays using FSUB1 and FSUB2 for Fusariumsubglutinans, FCORN2 and FVERT1 for F. verticillioides, and FCORN2 withFPRO1 for F. proliferatum amplified DNAs only from target species foreach PCR assay. No cross-reactivity with any of the other DNAs wasobserved. FSUB1 when used with FSUB2 in PCR reactions, when prepared asin Example 3, amplify only the isolates in Table 1 identified asFusarium subglutinans. Likewise, primers FCORN2 and FVERT1 amplifyproducts only with isolates identified as the target Fusariumverticillioides and primers FCORN2 and FPRO1 amplify from Fusariumproliferatum isolates only. No cross-reactivity is observed amongpreparations of non-target DNA from maize and other fungal pathogens.Furthermore, nonspecific amplification products are absent in allreactions performed.

Example 8 Use of Fusarium subglutinans, F. verticillioides, and F.proliferatum PCR Assays for Determination of Fungal Species Culturedfrom Field Samples

[0105] The maize ear rot PCR assays documented in the above examples areused to establish the speciation of unknown ear rot isolates culturedfrom field-grown maize in Stanton, Minn., USA (Table 2). PCRs areperformed as described in Example 3 using optimal primer pairs (FSUB1and FSUB2 for Fusarium subglutinans, FCORN2 and FVERT1 for F.verticillioides, and FCORN2 with FPRO1 for F. proliferatum) against DNAfrom the field isolates prepared as described in Example 1. Products arevisualized on an ethidium bromide stained gel. Results are scored aseither positive (+) or negative (−) for the amplification of target DNA.Any PCR product visible, of the correct size, is considered a positiveand nonspecifics are recorded if present. Results of each of these testsare shown in Tables 10 -12. TABLE 10 Results of F. subglutinans PCRAssays Against Isolates Collected from Field-grown Maize. F.subglutinans Isolate PCR Result Fm001 − Fm002 − Fm003 + Fm004 − Fm005 −Fm006 − Fm007 − Fm008 − Fm009 − Fm010 − Fm011 − Fm012 − Fm013 − Fm014 −Fm034 − Fm035 − Fm036 − Fm037 − Fm041 − Fm042 − Fm043 − Fm044 − Fm045 −Fm046 − Fm047 − Fm048 − Fm049 − Fm050 − Fm051 − Fm052 − Fm053 − Fm054 −Fm055 − Fm056 − BC3SO 189 − Fsub1 + Fsub2 + Fsub3 + Fsub4 +

[0106] Five of the forty-one isolates cultured from field-grown maizereact with the Fusarium subglutinans primers. TABLE 11 Results of F.proliferatum PCR Assays Against Isolates Collected from Field-grownMaize. F. proliferatum Isolate PCR Result Fm001 − Fm002 − Fm003 − Fm004− Fm005 − Fm006 − Fm007 − Fm008 − Fm009 − Fm010 + Fm011 − Fm012 − Fm013− Fm014 + Fm034 − Fm035 − Fm036 − Fm037A + Fm041 − Fm042 − Fm043 −Fm044A + Fm045 − Fm046 − Fm047A + Fm048 − Fm049 − Fm050 − Fm051 − Fm052− Fm053 − Fm054 − Fm055 − Fm056 − BC3SO 189 − Fsub1 − Fsub2 − Fsub3 −Fsub4 −

[0107] The Fusarium proliferatum specific primers react with five of theforty-one isolates cultured from field-grown maize. TABLE 12 Results ofF. verticillioides PCR Assay Against Isolates Collected from Field-grownMaize. F. verticillioides Isolate PCR Result Fm001 + Fm002 + Fm003 −Fm004 + Fm005 + Fm006 + Fm007 + Fm008 + Fm009 + Fm010 − Fm011 + Fm012 +Fm013 + Fm014 − Fm034 + Fm035 + Fm036 + Fm037 − Fm041 + Fm042 + Fm043 +Fm044 − Fm045 + Fm046 + Fm047 − Fm048 + Fm049 + Fm050 + Fm051 + Fm052 +Fm053 + Fm054 + Fm055 + Fm056 + BC3SO 189 − Fsub1 − Fsub2 − Fsub3 −Fsub4 −

[0108] Twenty-eight of the isolates cultured from field-grown maize wereidentified as Fusarium verticillioides with the species-specific PCRprimers FCORN2 and FVERT1. For the forty-one isolates tested, none reactwith more than one of the three tests. These experiments demonstrate theutility of the diagnostic PCR primers for characterizing isolates ofmaize ear rot.

Example 9 Use of Fusarium subglutinans, F. verticillioides, and F.proliferatum PCR Assays for Detection and Differentiation of FungalSpecies Infecting Husk Tissues Collected from Field-Grown Maize.

[0109] The maize ear rot PCR assays are used to establish the speciationof ear rot pathogens present in husk tissue samples taken fromfield-grown maize (Table 2). PCRs are performed as described in Example3 using FSUB1 and FSUB2 for Fusarium subglutinans, FCORN2 and FVERT1 forF. verticillioides, and FCORN2 with FPRO1 for F. proliferatum againstDNA from the field isolates prepared as in Example 2B. Products arevisualized on an ethidium bromide stained gel. Results are scored aseither positive (+) or negative (−) for the amplification of target DNA.Products are compared to a molecular size marker and positive controlson the gel to determine that the products scored are of the correct sizeand any nonspecific amplification products are recorded if present.Results of the Fusarium subglutinans test are shown in Table 13. TABLE13 Results of F. subglutinans Assay Against Various Maize Tissues SampleF. subglutinans Designation Tissue PCR Result H-5 Husk + H-9 Husk +SBP-2 Husk +

[0110] The three maize tissues tested are identified as positive for thepresence of Fusarium subglutinans target DNA. Fusarium proliferatum andF. verticillioides tests are also run against these husk tissues. Notarget DNA is detected in the maize tissues using the F. proliferatum orF. verticillioides assays. The results of these experiments show theutility of the maize ear rot assays in identifying and distinguishingspecies present in maize tissue samples without having to first culturethe organism out of the tissue. The primers in Example 6 can be used inPCR assays to directly characterize extractions of maize tissue.

Example 10 Determination of Primer Specificity to Purified FungalGenomic DNA Using MS 1 or MS2 primer Combinations

[0111] Primers MS1 and MS2 from the literature are designed to amplifymitochondrial small subunit rDNA. The MS 1 priming site lies upstream ofthe reverse primers FSUB2, FSUB3, FVERT1, FVERT2, and FPRO1. Using theconserved MS1 primer in combination with 3′ primers specific to a fungussuch as a Fusarium spp. in polymerase chain reactions performed as inExample 3 produces am assau ised tp detect the specific fungus. Forexample, MS 1 is combined with a 3′ primer listed in Table 5 such as:FSUB2 or FSUB3 to detect F. subglutinans; FVERT 1 or FVERT2 to detect F.verticillioides; and FPRO1 to detect F. proliferatum.

[0112] Similarly, the MS2 reverse primer in combination with 5′ primersspecific to a fungus such as Fusarium spp. are used to detect one ormore specific fungi in PCR reactions performed as in Example 3. Forexample, MS2 is combined with a 5′ primer listed in Table 5 such asFSUB1to detect F. subglutinants; and FCORN1 or FCORN2 to Fusarium spp. ingeneral Such an assay for Fusarium spp. could have utility in situationswhere detection of Fusarium spp. without differentiation of the speciespresent is desired.

[0113] While the present invention has been described with reference tospecific embodiments thereof, it will be appreciated that numerousvariations, modifications, and further embodiments are possible, andaccordingly, all such variations, modifications and embodiments are tobe regarded as being within the scope of the present invention.

[0114] Numerous patents, applications and references are discussed orcited within this specification, and all are incorporated by referencein their entireties.

1 24 1 682 DNA Fusarium verticillioides (syn. F. moniliforme) 1gctaacggct gaactggcaa cttggagaag tggcaagtct tccagtatgg ggagcaaaac 60agctatgggt caagtccgat atctttagga gaagtcttat tgtgagggcg agttttataa 120caccatagga ctggccgccc catatgaaaa gattatatta gaattgaatg aagctttgtt 180tatatattga taatgacagt atatatatcg tgtcttgact aattgcgtgc cagcagtcgc 240ggtaatacgt aagagactag tgttattcat cttaattagg tttaaagggt acccagacgg 300tcaatatagc ttataaaatg ttagtacttg actagagttt tatgtaagag ggcagtactt 360gaggaggaga gatgaaattt cgtgatacca aagggactct gtaaaggcga aggcagccct 420ctatgtaaaa actgacgttg aaggacgaag gcacagagaa caaacaggat tagataccca 480agtagtcttt gcagtaaatg atgaatgcca taggttagat gggtgggtta gtcgtagttg 540agttagttta gcaaactaat ggattcagac tagtccacca tatatttggt ctataaatga 600aagtgtaagc atttcacctc aagagtaatg tggcaacgca ggaactgaaa tcactagacc 660gtttctgaca ccagtagtga ag 682 2 689 DNA Fusarium proliferatum 2gctaacggct gaactggcaa cttggagaag tggcaagtct tccagtatgg ggagcaaaac 60agctatgggt caagtctgat atctttagga ggggcgaagc tcctcttatt gtgagggcga 120gttatataac accataggac tggccgcccc atatgaaaag attatattag aattgaatga 180agctttgttt atatattgat aatgacagta tatatatcgt gtcttgacta attgcgtgcc 240agcagtcgcg gtaatacgta agagactagt gttattcatc ttaattaggt ttaaagggta 300cccagacggt caatatagct tataaaatgt tagtacttga ctagagtttt atgtaagagg 360gcagtacttg aggaggagag atgaaatttc gtgataccaa agggactctg taaaggcgaa 420ggcagccctc tatgtaaaaa ctgacgttga aggacgaagg cacagagaac aaacaggatt 480agatacccaa gtagtctttg cagtaaatga tgaatgccat aggttagatg ggtgggctcg 540tctagttgag ttagtttagc aaactaatga tctagacgag cccaccgtat atttggtcta 600taaatgaaag tgtaagcatt tcacctcaag agtaatgtgg caacgcagga actgaaatca 660ctagaccgtt tctgacacca gtagtgaag 689 3 726 DNA gibberella zeae (syn.Fusarium graminearum) 3 gctaacggct gaactggcaa cttggagaag tggcaagtcttccagtatgg ggagcaaaca 60 gctatgggtc aagcccgata cctttaagag aagtcttattgtgagggcga gttgtataac 120 accatagggc tggccgcccc atatgaaaag attttattagaattgaatga aactttgttt 180 atatattgat aatgacagta tatatatcgt gtcttgactaattgcgtgcc agcagtcgcg 240 gtaatacgta agagactagt gttattcatc ttaattaggtttaaagggta cccagacggt 300 ctatatagct tataaaatgt tagtataaga ctagagttttatgtaagagg gcagtacttg 360 aggaggagag atgaaatttc gtgataccaa agggactctgtaaaggcgaa ggcagccctc 420 tatgtaaaaa ctgacgttga aggacgaagg cacagagaacaaacaggatt agatacccaa 480 gtagtctttg cagtaaatga tgaatgccat aggttagatctatatttcta ttataataat 540 acatttctat tatttattat aaaacgcatt ccttatatagcttcgcgcta taatatattt 600 tatatatagt gcagcagaaa tttttgtatc tggtctataaatgaaagtgt aagcatttca 660 cctcaagagt aatgtggcaa cgcaggaact gaaatcactagaccgtttct gacaccagta 720 gtgaag 726 4 690 DNA Fusarium subglutinans 4gctaacggct gaactggcaa cttggagaag tggcaagtct tccagtatgg ggagcaaaac 60agctatgggt caagtccgat atctttagga ggcgcgaagc tcctcttatt gtgagggcga 120gttttataac accataggac tggccgcccc atatgaaaag attatattag aattgaatga 180agctttgttt atatattgat aatgacagta tatatatcgt gtcttgacta attgcgtgcc 240agcagtcgcg gtaatacgta agagactagt gttattcatc ttaattaggt ttaaagggta 300cccagacggt caatatagct tataaaatgt tagtacttga ctagagtttt atgtaagagg 360gcagtacttg aggaggagag atgaaatttc gtgataccaa agggactcgg taaaggcgaa 420ggcagccctc taggtaaaaa ctgacgttga aggacgaagg cacagagaac aaacaggatt 480agatacccaa gtagtctttg cagtaaatga tgaatgccat aggttagatc tgagttggta 540gtctagttga gttagtttac taaactaatg atctatacaa gccagcctta gatttggtct 600ataaatgaaa gtgtaagcat ttcacctcaa gagtaatgtg gcaacgcagg aactgaaatc 660actagaccgt ttctgacacc agtagtgaag 690 5 522 DNA Fusarium subglutinans 5tccgttggtg aaccagcgga gggatcatta ccgagtttac aactcccaaa cccctgtgaa 60cataccaatt gttgcctcgg cggatcagcc cgctcccggt aaaacgggac ggcccgccag 120aggaccccta aactctgttt ctatatgtaa cttctgagta aaaccataaa taaatcaaaa 180ctttcaacaa cggatctctt ggttctggca tcgatgaaga acgcagcaaa atgcgataag 240taatgtgaat tgcagaattc agtgaatcat cgaatctttg aacgcacatt gcgcccgcca 300gtattctggc gggcatgcct gttcgagcgt catttcaacc ctcaagccca gcttggtgtt 360gggactcgcg agtcaaatcg cgttccccaa attgattggc ggtcacgtcg agcttccata 420gcgtagtagt aaaaccctcg ttactggtaa tcgtcgcggc cacgccgtta aaccccaact 480tctgaatgtt gacctcggat caggtaggaa tacccgctga ac 522 6 521 DNA Gibberellazeae 6 tccgttggtg aaccagcgga gggatcatta ccgagtttac aactcccaaa cccctgtgaa60 cataccttat gttgcctcgg cggatcagcc cgcgccccgt aaaaagggac ggcccgccgc 120aggaacccta aactctgttt ttagtggaac ttctgagtat aaaaaacaaa taaatcaaaa 180ctttcaacaa cggatctctt ggttctggca tcgatgaaga acgcagcaaa atgcgataag 240taatgtgaat tgcagaattc agtgaatcat cgaatctttg aacgcacatt gcgcccgcca 300gtattctggc gggcatgcct gttcgagcgt catttcaacc ctcaagccca gcttggtgtt 360gggagctgca gtcctgctgc actccccaaa tacattggcg gtcacgtcga gcttccatag 420cgtagtaatt tacacatcgt tactggtaat cgtcgcggcc acgccgttaa accccaactt 480ctgaatgttg acctcggatc aggtaggaat acccgctgaa c 521 7 534 DNA Fusariumproliferatum 7 tccgttggtg aaccagcgga gggatcatta ccgagtttac aactcccaaacccctgtgaa 60 cataccaatt gttgcctcgg cggatcagcc cgctcccggt aaaacgggacggcccgccag 120 aggaccccta aactctgttt ctatatgtaa cttctgagta aaaccataaataaatcaaaa 180 ctttcaacaa cggatctctt ggttctggca tcgatgaaga acgcagcaaaatgcgataag 240 taatgtgaat tgcagaattc agtgaatcat cgaatctttg aacgcacattgcgcccgcca 300 gtattctggc gggcatgcct gttcgagcgt catttcaacc ctcaagcccccgggtttggt 360 gttggggatc ggcgagccct tgcggcaagc cggccccgaa atctagtggcggtctcgctg 420 cagcttccat tgcgtagtag taaaaccctc gcaactggta cgcggcgcggccaagccgtt 480 aaacccccaa cttctgaatg ttgacctcgg atcaggtagg aatacccgctgaac 534 8 522 DNA Fusarium verticillioides (syn. F. moniliforme) 8tccgttggtg aaccagcgga gggatcatta ccgagtttac aactcccaaa cccctgtgaa 60cataccaatt gttgcctcgg cggatcagcc cgctcccggt aaaacgggac ggcccgccag 120aggaccccta aactctgttt ctatatgtaa cttctgagta aaaccataaa taaatcaaaa 180ctttcaacaa cggatctctt ggttctggca tcgatgaaga acgcagcaaa atgcgataag 240taatgtgaat tgcagaattc agtgaatcat cgaatctttg aacgcacatt gcgcccgcca 300gtattctggc gggcatgcct gttcgagcgt catttcaacc ctcaagccca gcttggtgtt 360gggactcgcg agtcaaatcg cgttccccaa attgattggc ggtcacgtcg agcttccata 420gcgtagtagt aaaaccctcg ttactggtaa tcgtcgcggc cacgccgtta aaccccaact 480tctgaatgtt gacctcggat caggtaggaa tacccgctga ac 522 9 19 DNA Artificialsequence misc_feature (1)..(19) Primer ITS1 9 tccgtaggtg aacctgcgg 19 1020 DNA Artificial sequence misc_feature (1)..(20) Primer ITS2 10gctgcgttct tcatcgatgc 20 11 20 DNA Artificial sequence misc_feature(1)..(20) Primer ITS3 11 gcatcgatga agaacgcagc 20 12 20 DNA Artificialsequence misc_feature (1)..(20) Primer ITS4 12 tcctccgctt attgatatgc 2013 21 DNA Artificial sequence misc_feature (1)..(20) Primer FCORN1 13gcaacttgga gaagtggcaa g 21 14 20 DNA Artificial sequence misc_feature(1)..(20) Primer FCORN2 14 aagtcttcca gtatggggag 20 15 20 DNA Artificialsequence misc_feature (1)..(20) Primer FSUB1 15 gtccgatatc tttaggaggc 2016 21 DNA Artificial sequence misc_feature (1)..(21) Primer FSUB2 16tcaactagac taccaactca g 21 17 21 DNA Artificial sequence misc_feature(1)..(21) Primer FSUB3 17 caaatctaag gctggcttgt a 21 18 20 DNAArtificial sequence misc_feature (1)..(20) Primer FVERT1 18 tggtggactagtctgaatcc 20 19 20 DNA Artificial sequence misc_feature (1)..(20)Primer FVERT2 19 tcaactacga ctaacccacc 20 20 22 DNA Artificial Sequencemisc_feature (1)..(22) Primer FPRO1 20 taaactaact caactagacg ag 22 21 19DNA Artificial sequence misc_feature (1)..(19) Primer FPRO2 21gatttcgggg ccggcttgc 19 22 18 DNA Artificial sequence misc_feature(1)..(18) Primer FPRO3 22 cgcaagggct cgccgatc 18 23 25 DNA Artificialsequence misc_feature (1)..(25) Primer MS1 23 cagcagtcaa gaatattagtcaatg 25 24 22 DNA Artificial sequence misc_feature (1)..(22) Primer MS224 gcggattatc gaattaaata ac 22

1-6. (canceled)
 7. A method for the detection of a fungal pathogen,comprising the steps of: (a) isolating DNA from a plant leaf infectedwith a pathogen; (b) subjecting said DNA to polymerase chain reactionamplification using a pair of primers wherein each primer has sequenceidentity with at least 10 contiguous nucleotides of a mitochondrialsmall subunit rDNA gene from from Fusarium subglutinans and wherein atleast one primer comprises the nucleotide sequence of SEQ ID NOS:13, 15or 16; and (c) detecting said fungal pathogen by visualizing the productor products of said polymerase chain reaction amplification. 8-12.(canceled)
 13. The method of claim 7, wherein the primers comprise: SEQID NO:15 and SEQ ID NO:16. 14-16. (canceled)
 17. A diagnostic kit usedin detecting Fusarium subglutinans comprising at least one primer havingthe nucleotide sequence of SEQ ID NO: 13, 15 or
 16. 18. A diagnostic kitused in detecting Fusarium proliferatum a fungal comprising a pair ofprimers of: SEQ ID NO:15 and SEQ ID NO:16.